1. Objective 2.1: Investigation of novel pathways regulating HSC self-renewal and differentiation Pathways involved in in vivo HSPC expansion. By exploiting the extensive HSPC amplification after transplantation, we aim to identify novel factors/pathways involved in HSPC self-renewal. We have performed experiments to identify conditions for optimal HSPC amplification after transplantation, including cell dose, timing of cell collection after transplantation, and the impact of serial transplantations. We found that lower cell doses (e.g. 1x106 cells) result in superior HSPC expansion in vivo compared to larger cell doses (e.g. 1x107 cells). The optimal timing for cell collection was 1 week; longer times in vivo (e.g. 2, 3, or 4 weeks) resulted in progressive differentiation to more mature progeny. Serial transplantations have led to engraftment levels too low to allow reliable detection and selection of HSPCs. In FY17, we compared the transcriptome of bona fide HSPCs (before transplant) and after transplant, using single cell RNA-Seq. Large dataset have been generated and analysis is ongoing. Several transcription factors involved in the maintenance of HSPC function have been identified and recent studies have suggested involvement of epigenetic mechanisms to orchestrate the activities of these factors to ensure blood homeostasis. DNA methylation of CpG dinucleotides is a key epigenetic modification that influences mammalian gene expression. CpG methylation is catalyzed by a family of DNA methyltransferase (DNMT) enzymes comprising three members, DNMT1, DNMT3a, and DNMT3b. Conditional ablation of DNMT3a in a mouse model resulted in progressive impairment of HSPC differentiation over serial transplantation, while simultaneously expanding HSPC numbers in the bone marrow. DNMT3a-null HSPCs upregulated HSPC multipotency genes and downregulated differentiation factors, and their progeny exhibited global hypomethylation and incomplete repression of HSC-specific genes. These data established DNMT3a as a critical participant in the epigenetic silencing of HSPC multipotency genes, thereby enabling efficient differentiation. In FY18, we intend to extend these findings by investigating whether transient downregulation of DNMT3a expression by CRISPR interference in human CD34+ cells may prevent differentiation of HSPCs and favor their self-renewal during short-term expansion ex vivo. 2. Objective 2.2: Potentiation of known and new pathways involved in HSC self-renewal with hypoxia. Activation of Notch signaling in human HSPCs by treatment with Notch ligand (Delta1) has enabled clinically relevant ex vivo expansion of short-term HSPCs in cord blood (CB) transplantation. However, expansion of long-term repopulating HSPCs has not been demonstrated with Delta1. Moreover, all gene therapy clinical trials targeting HSPCs have used adult CD34+ cells collected from either BM or cytokine-mobilized peripheral blood. Low O2 tension (hypoxia) plays a crucial role in adult HSPC regulation of self-renewal. A molecular link has been proposed in various stem/progenitor cell populations between hypoxia and Notch pathways, but their molecular interaction has not been investigated in human HSPCs. We hypothesized that Notch and hypoxia pathways could synergize to favor expansion of adult long-term repopulating HSPCs. G-CSF mobilized human CD34+ cells from 4 healthy subjects were cultured for 21 days in the presence of cytokines in hypoxia (1% O2) or normoxia (21% O2) in vessels coated with fibronectin alone or combined with an optimized concentration of immobilized Notch ligand (Delta1). The total number of CD34+ cells and CFUs expanded modestly with Delta1 in both normoxia and hypoxia. However, relative to uncultured cells, phenotypically defined HSCs increased significantly more with Delta1 in hypoxia compared to normoxia. Similarly, cells cultured with Delta1 in hypoxia resulted in increased human cell engraftment in NSG mice (29.3 11% human CD45+ cells,) compared to uncultured cells (7.0 0.1%) or cells cultured with Delta1 in normoxia (5.5 5.4%). Next, we performed limiting dilution analysis to measure the frequencies of long-term (LT) repopulating HSPCs within the CD34+ cell compartment at baseline and after 21 days in hypoxic or normoxic cultures supplemented with Delta1. LT-HSPCs in uncultured CD34+ cells were measured at the expected frequency (1 in 7,706; 95% CI of 3,446 to 17,232). When analyzed at 3 months post-transplantation, a limited (1.5-fold) increase in LT-HSPC frequency (1 in 5,090; 95% CI 2.456 to 10,550) was obtained from Delta1 normoxic cultures compared to uncultured cells. In contrast, the frequency of LT-HSPCs (1 in 1,586; 95% CI 680 to 3,701) was 5-fold higher in hypoxic Delta1 cultures compared to uncultured cells, and 4.2-fold higher than in normoxic Delta1 cultures. Studies for the expansion of genetically edited HSPCs using this approach are ongoing. Our data indicate that hypoxia potentiates Notch-induced expansion of human HSPCs and may be of benefit in stem cell transplantation and gene therapy applications. To confirm expansion of the most primitive long-term repopulating cells using the hypoxia-Delta1 platform, we have competitively transplanted rhesus macaques with HSPCs expanded in the presence of Delta1 under hypoxic or normoxic conditions. Results will be available in FY18. Evaluation of the molecular mechanisms underlying the intersection between Notch and hypoxia pathways is underway.